Organized Endothelial Cell Surface Signal Transduction in Caveolae Distinct from Glycosylphosphatidylinositol-anchored Protein Microdomains

doi:10.1074/jbc.272.11.7211

Organized Endothelial Cell Surface Signal Transduction in Caveolae Distinct from Glycosylphosphatidylinositol-anchored Protein Microdomains*

From the Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 and the
^‡ Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115

^§ To whom correspondence should be addressed:
Dept. of Pathology, Harvard Medical School, Research North-Beth Israel, 330 Brookline Ave., Boston, MA 02215
. Tel.: 617-667-3577; Fax: 617-667-3591; E-mail: jschnitz{at}bidmc.harvard.edu

Abstract

Regulated signal transduction in discrete microdomains of the cell surface is an attractive hypothesis for achieving spatial and temporal specificity in signaling. A procedure for purifying caveolae separately from other similarly buoyant microdomains including those rich in glycosylphosphatidylinositol-anchored proteins has been developed (Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J., and Oh, P. (1995) Science 269, 1435-1439) and used here to show that caveolae contain many signaling molecules including select kinases (platelet-derived growth factor (PDGF) receptors, protein kinase C, phosphatidylinositol 3-kinase, and Src-like kinases), phospholipase C, sphingomyelin, and even phosphoinositides. More importantly, two different techniques reveal that caveolae function as signal transducing subcompartments of the plasma membrane. PDGF rapidly induces phosphorylation of endothelial cell plasmalemmal proteins residing in caveolae as detected by membrane subfractionation and confocal immunofluorescence microscopy. This PDGF signaling cascade is halted when the caveolar compartment is disassembled by filipin. Finally, in vitro kinase assays show that caveolae contain most of the intrinsic tyrosine kinase activity of the plasma membrane. As signal transducing organelles, caveolae organize a distinct set of signaling molecules to permit direct regionalized signal transduction within their boundaries.

Footnotes

↵* This work was reported in part at the ASCB Annual Meeting in Washington, D.C. (Dec., 1995) and ASBMB/ASIP Joint Meeting in New Orleans (June, 1996) (FASEB J., 10, A998, 1996). This work was supported by NIH grant HL52766 (to J. E. S.) and was done during the tenure of an Established Investigator Award from the Am. Heart Assoc. and Genentech (to J. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TIM

Triton-insoluble membrane(s)

MDCK

Madin-Darby canine kidney

GPI

glycosylphosphatidylinositol

PDGF

platelet-derived growth factor

NRTK

nonreceptor tyrosine kinase

PKC

protein kinase C

Mes

2-(N-morpholino)ethanesulfonic acid

VEGF

vascular endothelial growth factor

PI 3-kinase

phosphatidylinositol 3-kinase

PI

phosphatidylinositol

PLC

phospholipase C

PAGE

polyacrylamide gel electrophoresis

DMEM

Dulbecco's modified Eagle's medium

IP₃

inositol 1,4,5-trisphosphate

RLMVEC

rat lung microvascular endothelial cell(s)

SM

sphingomyelin

P

plasma membrane

H

homogenate

T

Triton-soluble phase

V

vesicle.
↵² P. Oh, D. P. McIntosh, and J. E. Schnitzer, unpublished observations.
↵³ R. Campos, personal communication.
- Received October 16, 1996.
- Revision received December 9, 1996.